The lens of the eye is a complex organ that, with the cornea, focuses an image on the retina. Lens cells have glucose transporters, chloride channels, sodium and potassium channels, gap junctions, and many other standard transport proteins in common with the cells of other organs. Aquaporin zero (AQPO) however is a unique water channel protein found only in the fiber cells of the lens. This proposal examines the roles of AQPO using a combination of a biophysical measure of the water permeability induced by AQPO, molecular biology to design and construct informative mutants of AQPO and investigation of the effects of adding, altering or deleting AQPO in the lens of living animals. In this proposal we will investigate the role of AQPO in development and maintenance of the lens by disrupting its normal expression in the zebrafish (which has two AQPO genes which differ in their properties), by knocking down expression of AQPO genes using antisense morpholino oligos, by over expressing variant AQPO molecules under lens specific promoters or by combining these techniques (Aim 1). In parallel, we will investigate the molecular basis for the regulation of AQPO water permeability by calcium and hydrogen, two ions whose concentration varies considerably from the edge of the lens to its center, by expressing wild type and mutant cRNAs in Xenopus oocytes, then measuring the resulting permeability using a swelling assay. In particular we will examine cooperativity between monomers (Aim 2) and test the hypothesis the regulation of permeability by calcium is effected by a reversible, calcium-dependent block of half of the AQPO monomers in a tetra mer (Aim 3). Note: Though submitted as a revised abstract, the above is nearly identical to the abstract for our R01 AO application. This reflects our intention to pursue the same specific aims described in the previous proposal. We expect to make significant progress on all three specific aims, even in two years, but obviously we will not be able to carry the individual aims as far as would have hoped to do in five years.
Our study of AQP0 in the lens will in crease our understanding of the normal physiology of the lens and etiology of cataract, possibly leading to new approaches for treating or preventing cataract. It will also expand our understanding of how aquaporins in general are regulated and may provide a new approach to develop aquaporins as a target for drugs which could beneficially alter water permeability in such life threatening clinical situations as cerebral edema.
| Fields, James B; Németh-Cahalan, Karin L; Freites, J Alfredo et al. (2017) Calmodulin Gates Aquaporin 0 Permeability through a Positively Charged Cytoplasmic Loop. J Biol Chem 292:185-195 |
| Reichow, Steve L; Clemens, Daniel M; Freites, J Alfredo et al. (2013) Allosteric mechanism of water-channel gating by Ca2+-calmodulin. Nat Struct Mol Biol 20:1085-92 |
| Clemens, Daniel M; Németh-Cahalan, Karin L; Trinh, Lien et al. (2013) In vivo analysis of aquaporin 0 function in zebrafish: permeability regulation is required for lens transparency. Invest Ophthalmol Vis Sci 54:5136-43 |
| Hall, James E (2012) Through a glass darkly. EMBO Mol Med 4:1-2 |
